As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
All the references cited in this application are specifically incorporated by reference herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference:    1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;    2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia.
Delivery of molecules such as lipids, proteins, peptides, DNA, polysaccharides and/or combinations thereof (eg. lipopolysaccharides, lipoproteins), into cells is useful for a multitude of research and clinical purposes. For example, in order for researchers to study intracellular processes such as gene regulation and expression, DNA-protein interactions or protein-protein interactions, and so on, it is often essential to introduce molecules into cells, and desirable to do so with as high efficiency as possible. Currently researchers deliver molecules into cells, i.e. transfect cells, by a variety of means with variable efficiency. The efficiency of transfection of cells is dependent on a number of factors including cell type, rate and stage of cell division, and the individual properties of both the molecules to be transfected and the transfection reagent(s).
DNA vaccines are thought to elicit an immune response via uptake of DNA by antigen-presenting cells such as dendritic cells (DCs), which subsequently express the antigen encoded by the internalised DNA and present the antigen to the immune system as peptides in the context of MHC molecules. In small animal models, administration of DNA has been successful in inducing protective immune responses, but only low efficacies have been reported in human clinical trials, often requiring high doses of DNA to induce immune responses (Kutzler, M. A. & Weiner, D. B. 2004 J Clin Invest, 114(9), 1241-1244). Adenoviruses and retroviruses have been used as vectors for gene delivery, however concerns exist in relation to the safety of these vectors for human use (Buckley, R. H. 2002 Lancet, 360(9341), 1185-1186). To date, transfection of DCs with relatively safe, non-viral vectors has proven difficult.
DNA is a net negatively charged molecule. More specifically, the phosphate groups within the backbone of DNA are negatively charged. Therefore, cationic molecules, which have a net positive charge, can adsorb DNA via electrostatic interaction, and are potential carriers for DNA. Such cationic molecules include microparticles (Minigo, G. et al. 2007 Vaccine, 25(7), 1316-1327; Mollenkopf, H. J. et al. 2004 Vaccine, 22(21-22), 2690-2695), peptides (Gratton, J. P. et al. 2003 Nat Med, 9(3), 357-362; Riedl, P. et al. 2006 Methods Mol Med, 127, 159-169), or liposomes (Jiao, X. et al. 2003 Hepatology, 37(2), 452-460; Ewert, K. et al. 2002 J Med Chem, 45(23), 5023-5029).
Simply carrying DNA to antigen-presenting cells, however, is not sufficient enough to result in transfection, and in order to drive an antigen specific response, there must also be uptake of the DNA. Antigen-presenting cells of the immune system express toll-like receptors (TLRs) on their cell surface, which bind to a variety of ligands, largely derived from microorganisms. For example, TLR-2 is known to bind bacterial lipoproteins, TLR-4 is known to bind bacterial lipopolysaccharides, TLR-6, in association with TLR-1, is known to bind diacylated bacterial lipids, and TLR-9 binds to CpG DNA. Dendritic cell subsets have been shown to express no fewer than nine such TLRs. Engagement of one or more TLRs on the surface of DCs induces cell signalling pathways, which can lead to the maturation and activation of DCs, which is required for the induction of protective immunity.
The lipid moiety, dipalmitoyl-S-glyceryl cysteine (Pam2Cys), is a synthetic analogue of a bacterial lipoprotein known as MALP-2, derived from the cytoplasmic membrane of Mycoplasma fermentans. Pam2Cys is a ligand for both TLR-2 and TLR-6 (Okusawa, T. et al., Infect Immun 2004, 72(3), 1657-1665). Vaccines comprising Pam2Cys coupled to peptide epitopes can induce strong humoral and cellular responses. Engagement of TLR-2 by Pam2Cys coupled to peptide epitopes results in DC maturation, activation of transcription factors such as NF-κB, secretion of pro-inflammatory cytokines and eventual migration of DCs to the draining lymph nodes to activate epitope-specific naïve T cells (Jackson, D. C. et al. 2004 Proc Natl Acad Sci USA, 101(43), 15440-15445; Zeng, W. et al. 2002 J Immunol, 169(9), 4905-4912; Chua, B. Y. et al. 2007 Vaccine, 25(1), 92-101).